1. Field of Invention
This invention pertains to the art of methods for suppressing the invasion and spread of cancer cells and more particularly to the use of amiloride, alone or in combination with other agents, to inhibit the proliferation and invasive capability of epithelial based cancers which are dependent on the plasmin enzymatic cascade.
2. Background of the Invention
Cancer cells proliferate through a series of events such as separation of cells from the primary tumor mass, migration of the separated cells through solid tissue, penetration of the vascular system with embolization at a distant site (intravasation), escape of embolic cells from the confines of the capillary bed involved (extravasation), and parasitization of blood supply at the target site.
The mechanisms which allow these events to occur are perversions of normal physiologic processes and are dependent on a cascade of enzymatic activity which is initiated by the intracellular chemistry of the cancer cell. The trigger for this cascade is the activation of plasminogen to plasmin. This activation is a physiologic function which normally allows for the dissolution of blood clots within the vascular system, allows for the migration of blood cells through solid tissue to fight infection or to reject foreign material, and allows for the penetration of the ovum by the sperm cell.
In the cancer cell, large amounts of plasminogen activator, uPA, are produced. This activator binds to its own cell surface receptor sites. The surface-bound activator captures plasminogen from the circulatory system and converts it to the active form of plasmin. In turn, plasmin lyses the basement matrix, allowing the liberated cell to migrate through solid tissue. Plasmin also activates other tissue enzymes which dissolve connective tissue and the binding molecules of the cell wall of capillaries.
The end result of this complex activity is the local invasion of specific cancer cells as well as the capacity of those cells to invade the circulatory system and spread to distant sites. The types of cancer cells so affected are those derived from epithelial cells. These cancer types include the most commonly found in man, such as breast, colon, stomach and lung.
One trigger mechanism for the enzymatic cascade is the activation of plasminogen to plasmin by the specific activating enzyme Urokinase Plasminogen Activator or uPA. The capacity of the cancer cell to invade and spread may be depressed by inhibition of this trigger mechanism.
One substance which has been found to inhibit the activator, uPA, is a acylguanidine, known under the proprietary name, Amiloride. This drug is a potassium sparing diuretic which has been marketed for years for the purpose of controlling hypertension. It is used most often in conjunction with more powerful diuretics. Amiloride prevents the loss of potassium which occurs with those diuretic agents.
In 1988, an article by J. A. Kellen et. al. (Antimetastatic Effect of Amiloride in an Animal Tumor Model, Anticancer Res, 8, 1373-1376) presents a study using Fisher rats inoculated with breast cancer cells. The in vivo study showed that continuous administration of Amiloride via drinking water prevented the formation of lung cancer in the study animals.
The present invention confirms the suppression of experimentally induced metastases through the use of amiloride. In addition, a time and dose-dependency relationship of amiloride administration is demonstrated in the present invention.
Because known inhibitors in the art will suppress, but not eliminate the invasive capability of the cancer cells, one may surmise that more than the above-mentioned trigger mechanism in the enzymatic cascade should be targeted. A combination of inhibitors which attack separate parts of the chain reaction forms a part of the present invention.
In addition to the use of amiloride alone, the present invention is directed to the combination of the action of amiloride, which is a uPA inhibitor, with a metalloprotease inhibitor such as Batimistat which is a proprietary name of British Biotech, and/or hydrochlorothiazide, which promotes the excretion of potassium which could be retained by the other inhibitors.